Automatic determination of urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid by liquid chromatography with electrochemical detection

An automated liquid chromatographic method for the determination of urinary concentrations of 4-hydroxy-3-methoxymandelic acid (VMA) is described. Urine samples are purified by solid-phase extraction on an anion-exchange cartridge and automated on-line chromatographic elution is carried out using a Varian AASP (advanced automated sample processor) system. The column effluent is monitored with an electrochemical detector using a glassy carbon working electrode. The method allows the determination of VMA in 0.05 ml of normal urine with a relative standard deviation of less than 3%. The analysis time can be shortened by use of back-flushing technique, and the correlation with a classical (but non-automated) VMA analysis method is excellent.     

Systematic error in automated in-tube solid-phase microextraction

The widely employed configuration for automated in-tube solid-phase microextraction (SPME) involves modification of a commercial liquid chromatographic autosampler into an automated extraction device. This popular configuration is demonstrated to result in an inherent systematic error in the quantitation of analyte in a given matrix. The source of error is traced to the accumulation of analyte in the extraction and the pre-extraction segment (i.e., sample loop, metering valve and tubing prior to the metering valve) of the autosampler where the analyte comes in contact with the residual mobile phase. This results in cross-contamination due to sample/mobile phase mixing. The quantity of analyte accumulated in these segments is shown to consistently increase with the increasing number of draw/eject cycles. As a result of the accumulation, the amount of analyte recorded leads to inaccurate quantitative information, leading to overestimation of the limit of detection and limit of quantitation, when automated in-tube SPME is employed as an approach for sample enrichment. Insertion of a 100-microl air plug prior to extraction step was able to significantly minimize sample/mobile phase mixing of analyte with the residual mobile phase in the pre-extraction and extraction step, thus minimizing the systematic error.     

Fully automated high-performance liquid chromatographic assay for the analysis of free catecholamines in urine

A totally automated and reliable high-performance liquid chromatographic method is described for the routine determination of free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines were isolated from urine samples using small alumina columns. A standard automated method for pH adjustment of urine before the extraction step has been developed. The extraction was performed on an ASPEC (Automatic Sample Preparation with Extraction Columns, Gilson). The eluate was collected in a separate tube and then automatically injected into the chromatographic column. The catecholamines were separated by reversed-phase ion-pair liquid chromatography and quantified by fluorescence detection. No manual intervention was required during the extraction and separation procedure. One sample may be run every 15 min, ca. 96 samples in 24 h. Analytical recoveries for all three catecholamines are 63-87%, and the detection limits are 0.01, 0.01, and 0.03 microM for norepinephrine, epinephrine and dopamine, respectively, which is highly satisfactory for urine. Day-to-day coefficients of variation were less than 10%.     

On-line coupling of extraction with gas chromatography

A recent trend in the development of analytical methodologies is the integration of the sample preparation step directly with the chromatographic system. In integrated on-line systems, extraction, clean-up, separation and detection are connected with each other and the whole analytical procedure takes place in a closed, usually automated system. In this review, a critical overview of the principles and benefits of on-line coupling of extraction with gas chromatography is presented. Techniques suitable for the extraction and analysis of various types of compounds in both solid and liquid samples are presented. The potential of on-line techniques is discussed and illustrated with selected examples.     

Automated sample preparation using supported liquid membranes for liquid chromatographic determination of sulfonylurea herbicides

Summary Sample preparation for determination of sulfonylurea herbicides in aqueous samples is investigated. The technique studied utilizes extraction and back extraction in an automated flow system and is coupled on-line to a liquid chromatographic system. The extraction unit consists of an immobilized liquid membrane, separating two aqueous phases. From the acidified donor phase the analytes are extracted into the organic solvent of the membrane. After traversing the membrane they are back extracted into an alkaline/neutral aqueous acceptor phase. They are trapped in the acceptor by dissociation, making them insoluble in the membrane.Studies of the sample preparation system concern factors like channel length of separators, distribution coefficients of analytes and use of a precolumn instead of loop for chromatographic injections. Effects of the internal diameter of the analytical column as well as the detection of the sulfonylurcas are investigated.     

Chromatographic determination of vitamins in foods.

Chromatographic methods for the determination of water- and fat-soluble vitamins in foods are reviewed. For each vitamin, sample preparation, detection problems and chromatographic conditions are presented and discussed. High-performance liquid chromatography (HPLC) is becoming a standard method in vitamin assay, especially for routine work. HPLC systems can be automated using in-line solid-phase extraction and column switchings, resulting in very sensitive methods, even when simple UV detection is employed.     

An ion chromatographic separation method for the sequential determination of <Superscript>90</Superscript>Sr, <Superscript>241</Superscript>Am and Pu isotopes in a urine sample

A radiobioassay method has been developed for the sequential determination of ⁹⁰Sr, ²⁴¹Am and Pu isotopes in a urine sample. Unlike the existing methods using multiple extraction chromatographic cartridges, this work demonstrates an application of an automated ion chromatographic (IC) system for the separation of these radionuclides on a single IC column. The method meets the bioassay performance criteria for relative bias and relative precision as recommended by ANSI/HPS N13.30-2011. The detection limits for the radionuclides are found to be satisfactory for medical intervention in case of an accidental exposure scenario. Sample preparation time is less than 11 h.     

A robotic sample preparation scheme for the high performance liquid chromatographic determination of ivermectin in animal plasma

A lengthy sample preparation scheme for the high performance liquid chromatographic determination of the antiparasitic agent ivermectin at ppb concentrations in animal plasma is adapted to a laboratory robotic system. Sample treatment involves both liquid-liquid partitioning and solid phase extraction. Specific modifications to the manual procedure include the use of serial vortex mixings in place of batchwise lateral shaking and the substitution of small (6 mL), disposable solid phase extraction columns driven by compressed gas for large (25 mL), gravity-fed, reusable glass columns. Coupling these columns to an automated solvent dispensing device simplifies handling of the large solvent volumes prescribed in the manual procedure. A productivity gain over the manual procedure is realized when operating the robotic system in the single sample mode, and an additional gain is achieved by integrating the system for multiple sample handling. Equivalence to the manual procedure is demonstrated.     

Profiling of impurities in illicit amphetamine samples by high-performance liquid chromatography using column switching

A simple high-performance liquid chromatographic method, suitable for routine profiling of impurities in illegally produced amphetamine, has been developed. Amphetamine is dissolved in acetonitrile-citrate buffer (pH 3) (2:8) and injected directly without further sample pre-treatment. The impurities are enriched on-line on a C8 extraction column, while amphetamine and polar diluents are washed out with water. After washing for 1.5 min, a six-port valve is switched and an acetonitrile-0.2 M butylamine in water (pH 8) gradient elutes the impurities from the extraction column on to a C18 analytical column where they are separated. The compounds are monitored by UV detection at 220 and 254 nm. The total extraction and analysis time is 30 min. The method allows automated extraction and analysis to be performed.     

Coupling of biological sample handling and capillary electrophoresis.

The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) requires efficient sample preparation (i.e., concentration and clean-up) procedures to remove interfering solutes (endogenous/exogenous and/or low-/high-molecular-mass), (in)organic salts and particulate matter. The sample preparation modules can be coupled with CE either off-line (manual), at-line (robotic interface), on-line (coupling via a transfer line) or in-line (complete integration between sample preparation and separation system). Sample preparation systems reported in the literature are based on chromatographic, electrophoretic or membrane-based procedures. The combination of automated sample preparation and CE is especially useful if complex samples have to be analyzed and helps to improve both selectivity and sensitivity. In this review, the different modes of solid-phase (micro-) extraction will be discussed and an overview of the potential of chromatographic, electrophoretic (e.g., isotachophoresis, sample stacking) and membrane-based procedures will be given.     

Membrane-based techniques for sample enrichment

Sample preparation techniques based on non-porous membrane extraction generally offer a high degree of selectivity and enrichment power, together with convenient possibilities for direct and automated connections to chromatographic and other analytical instruments. In this review principles and applications for techniques as supported liquid membrane extraction, microporous membrane liquid-liquid extraction, polymeric membrane extraction and membrane extraction with a sorbent interface are described and compared.     

Automated microanalysis of carbamazepine and its epoxide and trans-diol metabolites in plasma by column liquid chromatography.

A fully automated high-performance liquid chromatographic procedure for the simultaneous determination of carbamazepine and its main metabolites, epoxycarbamazepine and dihydroxycarbamazepine, in plasma is described. Liquid-solid extraction on disposable C18 columns and reversed-phase chromatography on a 3 microns particle size C18 column were combined and automated by using the Automatic Sample Preparation with Extraction Columns system. Ultraviolet detection was performed at 210 nm. 5,6-Dihydro-11-oxo-11H-dibenz[b,e]azepine-5-carboxamide was used as internal standard. A small plasma volume (100 microliters) was required. The total run time for the assay of one sample was about 10 min. The assay demonstrated good reproducibility. The limit of quantitation was 0.1 mumol/l (about 25 ng/ml).     

Extraction of Nonylphenol and Nonylphenol Ethoxylates from River Sediments: Comparison of Different Extraction Techniques

Summary Five different extraction techniques (Soxhlet, automated Randall, accelerated solvent extraction, microwave-assisted solvent extraction and extraction with a surfactant solution) have been evaluated for the determination of nonylphenol (NP) and nonylphenol ethoxylates (NPEO) in river sediments. All the techniques were applied to the same three samples collected from northern Italian rivers. The analyses were performed with two RP columns, with different stationary reversed phases—a classical C₁₈ phase and a hexyl–phenyl phase. The recoveries and reproducibility of the different extraction techniques were comparable and all the methods gave reliable results. The variance of the results was dominated by the variance in sample homogeneity, sample preparation, and chromatographic analysis. A choice between the methods can be made on the basis of the cost and safety of each technique. Preliminary results obtained from use of a water-based extraction method with a surfactant solution (Tween-80), and its application to analysis of sediment and of worm tissue, are also presented.Presented at: Chemical Analysis and Risk Assessment of Emerging Contaminants, Barcelona, Spain, November 28–30, 2002     

Determination of four fluoroquinolones in milk by on-line immunoaffinity capture coupled with reversed-phase liquid chromatography.

An automated, on-line immunoaffinity extraction method was developed for the analysis of 4 fluoroquinolones in milk: ciprofloxacin, difloxacin, enrofloxacin, and sarafloxacin. This method involves analyte extraction using an immunoaffinity capture column containing anti-fluoroquinolone antibodies coupled on-line with reversed-phase column chromatography. Liquid chromatographic analyses were performed by isocratic elution using a mobile phase of 2% acetic acid-acetonitrile (85 + 15) and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 278 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Recoveries from fortified raw milk samples (5-50 ppb of each fluoroquinolone) ranged from 72 to 90%, with standard deviations of < or = 8%.     

Rapid and high-throughput determination of cationic surfactants in environmental water samples by automated on-line polymer monolith microextraction coupled to high performance liquid chromatography-mass spectrometry

A rapid, high throughput and sensitive method was presented for automated determination of cationic surfactants in environmental water samples. The method was based on an automated analysis platform that was composed of on-line polymer monolith microextraction (PMME) and high performance liquid chromatography-mass spectrum (HPLC-MS) with an autosampler. A poly(methacrylic acid-co-ethylene dimethacrylate) (MAA-co-EDMA) monolith was selected as the sorbent for purification and enrichment of cationic surfactants in environmental water samples while a new mixed-mode chromatographic column packed with octyl and sulfonic acid co-bonded silica (OSS) was employed for separation and quantitative determination of cationic surfactants in water samples. By integrating sample preparation, chromatographic separation and MS detection into one automated platform, it makes the whole analysis procedure simple, accurate, and time and labor-saving. Several parameters affecting the extraction performance were investigated. Under the optimized conditions, the proposed method was applied to the analysis of seven cationic surfactants in environmental water samples. Good linearities were obtained for all cationic surfactants with R2 larger than 0.9895. The limits of detection were found to be in the range of 15-24 ng/L. The method recoveries of the cationic surfactants spiked in water samples were from 80.5% to 115.1%, with relative standard deviations less than 12.4%.     

High-throughput quantitative bioanalysis by LC/MS/MS

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.     

Fast systematic approach for the determination of drugs in biological fluids by fully automated high-performance liquid chromatography with on-line solid-phase extraction and automated cartridge exchange. Application to cebaracetam in human urine.

A fast stepwise systematic approach for the conversion of conventional reversed-phase high-performance liquid chromatographic (HPLC) assays involving liquid-liquid extraction of biological fluids into fully automated HPLC assays using solid-phase extraction and cartridge exchange is described. The suitability of this procedure is demonstrated for the determination of cebaracetam in human urine.     

A review of solid phase extraction: Basic principles and new developments

Summary A review with 178 references on the basic principles and recent developments in the solid phase extraction is presented. New solid phases, chromatographic modes, experimental configurations and off-line and on-line automated devices are discussed.     

Planar solid phase extraction--a new clean-up concept in multi-residue analysis of pesticides by liquid chromatography-mass spectrometry

Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography coupled to mass spectrometry. As a completely new approach, highly automated planar chromatographic tools were applied for powerful clean-up, called high-throughput planar solid phase extraction (HTpSPE). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC-MS interface. HTpSPE resulted in extracts nearly free of interference and free of matrix effects, as shown for seven chemically representative pesticides in four different matrices (apples, cucumbers, red grapes, tomatoes). Regarding the clean-up step, quantification by LC-MS provided mean recovery (against solvent standards) of 90-104% with relative standard deviations of 0.3-4.1% (n=5) for two spiking levels of 0.1 and 0.5 mg/kg. Clean-up of one sample was completed in a manner of minutes, while running numerous samples in parallel at reduced costs, with very low sample and solvent volumes.     

A Total Solution to Baseline Separation of 20 Brominated Flame Retardant Additives in Electronic Products with Automated Soxhlet Hot Extraction and Gas Chromatography‐Mass Spectrometry

An efficient and reliable separation technique based on automated Soxhlet hot extraction (AHSE) was developed and validated. It can be applied to rapid separations of 20 persistent organic pollutants, including two types of brominated flame retardants (BFRs), polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs) contained in nonmetallic component parts of electronic products. The qualitative chromatographic analyses were carried out by using a gas chromatography coupled with a mass spectrometry detector (GC‐MS). The 20 persistent organic pollutants were simultaneously and completely separated by a 15 meter HP‐5MS short capillary column in 25 minutes. Through the tests of extraction performance, effects of solvent and extraction time on selected BFRs were investigated; toluene and 120 min extraction time were chose as the optimum conditions. Besides, this article examines the influence of temperature on the chromatographic analysis, the optimum temperature parameters were 280 °C and 320 °C for injector and column, respectively. The ASHE‐GCMS method was validated for the analysis of the certified reference material of CRM8110‐a and IRMM310. The limits of detection (LOD) for polymer sample was 0.55‐4.50 μg mL^?1; linearity range from 0.11 to 16 μg mL^?1. The proposed methodology can fully meet the requirement of relational directives.